Misfolded protein sensor method

ABSTRACT

A catalytic conformational sensor method for detecting abnormal proteins and proteinaceous particles. The method is based on the interaction of a peptide fragment or probe with an abnormal proteinaceous particle. The interaction catalyzes transformation of the probe to a predominately beta sheet conformation and allows the probe to bind to the abnormal proteinaceous particle. This in turn, catalyzes propagation of a signal associated with the test sample-bound probe. As a result signals can be propagated even from samples containing very low concentrations of abnormal proteinaceous particles. The peptide probes can be designed to bind to a desired peptide sequence or can even be based on dendrimer structure to control further aggregate propagation.

RELATED ART

This document claims priority of U.S. provisional patent applicationSer. No. 60/295,456 filed on May 31, 2001, with respect to subjectmatter therein; said provisional application fully being incorporatedherein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The inventions disclosed herein were partly funded by grants. Therefore,to the extent that rights to such inventions may accrue to the U.S.Government, the following statement, required under 37 C.F.R.§401.14(f)(4) applies: This invention was made with government supportunder National Institutes of Health (NIH) Grant number is 5 R44HL070399-04. The government has certain rights in the invention.

BACKGROUND 1. Field of the Invention

This invention relates generally to a catalytic conformational sensormethod and application of such method for detecting proteins andproteinaceous particles; and more particularly to detecting misfolded ordisease-associated proteins and proteinaceous particles.

The present invention detects misfolded or abnormal conformations ofproteins or peptides such as those contributing to “folding diseases”.The “folding diseases” are characterized by proteins with destabilizingconformers which tend to aggregate and eventually form toxic plaques inbrain and other tissue. See Bucciantini, M., et al. (2002) InherentToxicity of Aggregates Implies a Common Mechanism for Protein MisfoldingDiseases. Nature 416:507-511.

These “folding diseases” can be hard to diagnose since the diseasesymptoms may be latent where the aggregates are slowly building up overtime and go through stages of increased aggregation leading to fibrilformation and eventual plaque deposition leading to impairment ofcellular viability. Such misfolding of peptides and aggregate formationis believed to play a key role in Alzheimer's disease where beta-amyloidprotein (or A beta, a 39-42 residue peptide) forms fibrillar depositsupon a conformer change; Huntington's disease where insoluble proteinaggregates are formed by expansion of poly-glutamine tracts in theN-terminus of huntingtin (Htt), an antiapoptotic neuronal protein; andnoninfectious cancers such as in cases where tumor-associated cellsurface NADH oxidase (tNOX) has prion-like properties such asproteinase^(R), ability to form amyloid filaments and the ability toconvert the normal NOX protein into tNOX. See Kelker, et al.Biochemistry (2001) 40:7351-7354. for more information on tNOX.

The present invention, however, is not limited to the detection ofproteins or peptides in folding-disease or infectious samples. It alsoincludes detection of proteinaceous particles such as prions. Prions aresmall proteinaceous particles with no nucleic acids, thus are resistantto most nucleic-acid modifying procedures and proteases. The normalprion (PrP) protein is a cell-surface metallo-glyroprotein that ismostly an alpha-helix and loop structure as shown in FIG. 8, and isusually expressed in the central nevrvous and lymph systems. It'sproposed function is that of an antioxidant and cellular homeostasis.

The abnormal form of the PrP, however, is a conformer which is resistantto proteases and is predominantly beta-sheet in its secondary structureas shown in FIG. 9. It is believed that this conformational change insecondary structure is what leads to the aggregate and eventualneurotoxic plaque deposition in the prion-disease process.

The abnormal prion are infectious particles that play key roles in thetransmission of several diseases such as Creutzfeldt-Jakob syndrome,chronic wasting disease (CWD), nvCJD, transmissible spongiformencephalopathy (TSE), Mad Cow disease (BSE) and scrapie a neurologicaldisorder in sheep and goats¹. ¹ Clayton Thomas, Tabor's CyclopedicMedical Dictionary (Phil., F. A. Davis Company, 1989), at 1485.

Diseases caused by prions can be hard to diagnose since the disease maybe latent where the infection is dormant, or may even be subclinicalwhere abnormal prion is demonstrable but the disease remains an acute orchronic symptomless infection. Moreover, normal homologues of aprion-associated protein exist in the brains of uninfected organisms,further complicating detection.² Prions associate with a proteinreferred to as PrP 27-30, a 28 kdalton hydrophobic glycoprotein, thatpolymerizes (aggregates) into rod-like filaments, plaques of which arefound in infected brains. The normal protein homologue differs fromprions in that it is readily degradable as opposed to prions which arehighly resistant to proteases. Some theorists believe that prions maycontain extremely small amounts of highly infectious nucleic acid,undetectable by conventional assay methods.³ As a result, many currenttechniques used to detect the presence of prion-related infections relyon the gross morphology changes in the brain and immunochemistrytechniques that are generally applied only after symptoms have alreadymanifest themselves. Many of the current detection methods rely onantibody-based assays or affinity chromatography using brain tissue fromdead animals and in some cases capillary immunoelectrophoresis usingblood samples. ² Ivan Roitt, et al., Immunology (Mosby-Year Book EuropeLimited, 1993), at 15.1.³ Benjamin Lewin, Genes IV (Oxford Univ. Press,New York, 1990), at 108.

The following is an evaluation of current detection methods.

-   -   Brain Tissue Sampling. Cross-sections of brain can be used to        examine and monitor gross morphology changes indicative of        disease states such as the appearance of spongiform in the        brain, in addition to immunohistochemistry techniques such as        antibody-based assays or affinity chromatography which can        detect disease-specific prion deposits. These techniques are        used for a conclusive bovine spongiform encephalopathy (BSE)        diagnosis after slaughter of animals displaying clinical        symptoms. Drawbacks of tissue sampling include belated detection        that is possible only after symptoms appear, necessary slaughter        of affected animals, and results that takes days to weeks to        complete.    -   Prionic-Check also requires liquified-brain tissue for use with        a novel antibody under the Western Blot technique. This test is        as reliable as the immunochemistry technique and is more rapid,        yielding results in six to seven hours, but shares the drawbacks        of the six-month lag time between PrP^(S) accumulation        (responsible for the gross morphology changes) in the brain and        the display of clinical symptoms, along with the need for        slaughter of the animal to obtain a sample.    -   Tonsillar Biopsy Sampling. Though quite accurate, it requires        surgical intervention and the requisite days to weeks to obtain        results.    -   Body Fluids: Blood and Cerebrospinal Sampling. As in the above        detection methods, results are not immediate    -   Electrospray ionization mass spectrometry (ESI-MS), nuclear        magnetic resonance NMR, circular dichroism (CD) and other        non-amplified structural techniques. All of these techniques        require a large amount of infectious sample, and have the        disadvantage of requiring off-site testing or a large financial        investment in equipment.

The following is a survey of currently approved and certified EuropeanUnion (EU) prion-detection tests.

-   -   Prionics—in Switzerland. The test involves Western blot of        monoclonal antibodies (MABs) to detect PrP in brain tissue from        dead animals in seven to eight hours.    -   Enfer Scientific—in Ireland. The test involves ELISA-based        testing on spinal cord tissue from dead animals in under four        hours.    -   CEA—in France. The test involves a sandwich immunoassay using        two monoclonals on brain tissue collected after death in under        twenty-four hours.

The EU Commission's evaluation protocol has sensitivity, specificity anddetection limits and titre. The sensitivity of a test is the proportionof infected reference animals that test positive in the assay. Itpreviously used 300 samples from individual animals to assess thiselement. The specificity of a test is the proportion of uninfectedreference animals that test negative in the assay. Previously used 1,000samples from individual animals for this purpose. In order to testdetection limits, various dilutions ranging from 10⁰ to 10⁻⁵ of positivebrain homogenate were used. A table showing an evaluation of EU testresults is shown in FIG. 12. Even with high degrees of sensitivity andspecificity, however, the fact remains that these tests must beperformed post-mortem and require working with large amounts of highlyinfectious biohazard materials.

The Center for Disease Control (CDC) classifies prions as Risk Group 2agents requiring Biosafety Level 2 (BSL2) containment. As a result manyof the above operations are carried out under BSL2 physical containmentwith elevated safety practices more typical of a BSL3 lab. Prions can beinactivated by fresh household bleach, 1 molar NaOH, 4 molar guanidinereagents, or phenol followed by 4.5 hours of autoclaving at 132° C.Procedures involving brain tissue from human patients with neurologicaldegenerative disorders pose special challenges and should be handledwith the same precautions as HIV+ human tissue. Thus, working with largeamounts of such biohazardous materials can be an obstacle to quick andsimple testing of mass quantities or assembly-line samples as well ascumbersome even for small applications.

In addition to working with relatively large amounts of biohazardousmaterials and taking several hours to weeks for detection, many of theprior art methods have the added difficulty that they are performed postmortem.

As can now be seen, the related art remains subject to significantproblems, and the efforts outlined above—although praiseworthy—have leftroom for considerable refinement. The present invention introduces suchrefinement.

SUMMARY OF THE DISCLOSURE

The present invention is based on the interaction between lowconcentration levels of abnormal proteinaceous particles and a peptidefragment or probe to induce transformation and propagation of the probebound to the abnormal proteinaceous particles initially present within atest sample. Thus, in a preferred embodiment, infectious levels of atest sample can be propagated even from low concentrations.

The present invention uses catalytic propagation to exploitconformational changes in proteins associated with a particular diseaseprocess, such as transmissible spongiform encephalopathy (TSE).Catalytic propagation basically amplifies the number of existing proteinfragments causing aggregates to form. The aggregates of conformationallychanged protein fragments are then easily detected using commonanalytical techniques.

As a result, the present invention allows testing to be done using rapidand cost-effective analytical techniques, even on, heretofore difficultto detect, small sample sizes and is widely applicable to tissues andbody fluids other than those found in brain. Results of the presentinvention can easily and immediately interpreted using familiaranalytical instrumentation. Additionally, the present invention canamplify a weak signal, thus can be successfully applied to small or weaksamples such as those associated with body fluids; thereby opening thedoor to analysis of tissues and fluids for the elusive diseasesdiscussed above. Moreover, this allows the method to be relativelynoninvasive in that it does not need to be performed post-mortem; andbecause it does not need to be performed post-mortem it can be appliedto presymptomatically.

The foregoing may be a description or definition of the first facet oraspect of the present invention in its broadest or most general terms.Even in such general or broad form, however, as can now be seen thefirst aspect of the invention resolves the previously outlined problemsof the prior art.

Because the present invention allows detection using samples with verylow levels of infectious agents and involves amplifying a peptide probeas opposed to a whole potentially infectious protein, many of theprevious biohazard-handling concerns are reduced.

Now turning to another of the independent facets or aspects of theinvention: in preferred embodiments of this facet, the peptide probesare designed for the detection of a desired sequence and so haveadaptable levels of selectivity and specificity built into the method.Also, intrinsic optical fluors such as pyrene can be designed into thepeptide probe allowing simple, single step optical detection of theabnormal proteinaceous particles.

All of the foregoing operational principles and advantages of thepresent invention will be more fully appreciated upon consideration ofthe following detailed description, with reference to the appendeddrawings, of which:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a pictoral representation of conformers of transmissiblespongiform encephalopathies (TSE) and probes in the form of labeledpeptides and labeled dendrimers;

FIG. 2 is a pictoral representation of TSE protein detection schema;

FIG. 3 is a graph showing the conformational changes associated with apoly-L-lysine test peptide using circular dichroism;

FIG. 4 is a graph comparing the circular dichroism results of thepoly-L-lysine test peptide at different temperatures and pH;

FIG. 5 is a table comparing the circular dichroism results of thepoly-L-lysine test peptide at different temperatures and pH;

FIG. 6 is a graph of data for fluorescence resonance energy transfer(FRET) experiments for proximal and distal locations in an α-helicalbundle structure undergoing conformational change;

FIG. 7 is a graph of the driving force necessary to overcome the energydifference between two different

FIG. 8 is a structural diagram of a normal PcP protein, a cell-surfacemetallo-glycoprotein that is expressed in the central nervous andlymphatic systems, and that is characterized as having mostly analpha-helix and loop structure;

FIG. 9 is a structural diagram of the PcP protein that has shifted to apredominately beta structure in which it is likely for form aggregatesand neurotoxic fibrils eventually leading to plaque deposition;

FIG. 10 is a pictoral representation of amplification of signal andpropagation of conformational change without increased aggregation bythe addition of dendrimers of the invention to a test sample;

FIG. 11 is a structural diagram of proteins used in the current priorart prion-diagnostic market; wherein FIG. 11 a on the left shows thePrPsens protein molecule and FIG. 11 b on the right shows a PrPresprotein molecule;

FIG. 12 is a table evaluating the current prior art in European Unioncertified prion-diagnostic tests

FIG. 13 is a comparison showing selected PrP sequences among sixdifferent species, i.e., Seq. Id. No. 1 through Seq. Id. No. 6;

FIG. 14 shows peptide sequences for the synthetic peptide probes 19-merSeq. Id. No. 7, and 14-mer, Seq. Id. No. 8;

FIG. 15 is a graph of fluorescence detection experimental resultsshowing the effects of peptide concentration;

FIG. 16 is a graph of fluorescence detection experimental resultsshowing the effects of peptide concentration likely showing excimeremission at approximately 460 nanometers (nm);

FIG. 17 is a graph of fluorescence detection experimental resultsshowing pyrene's excitation of fluorescence;

FIG. 18 is a graph of fluorescence detection experimental resultsshowing pyrene's excitation spectra for fluorescence at 398 andapproximately 460 nm;

FIG. 19 is a graph comparing the circular dichroism results of severalpeptides ranging in concentration from 20 to 100 milli Molar (mM) undervarying buffer conditions;

FIG. 20 is a graph comparing the circular dichroism results of severalpeptides including the synthetic peptides of Seq. Id. No. 7 and Seq. Id.No. 8 under varying buffer conditions;

FIG. 21 shows experimental results of the conformational lability of thesynthetic peptides. FIG. 21 a on the left show that 14-mer, Seq. Id. No.8, assumes a beta-sheet conformer while the longer analog, 19-mer, Seq.Id. No. 7, remains coiled. FIG. 21 b on the right shows that addition of14-mer, Seq. Id. No. 8, to 19-mer, Seq. Id. No. 7, initiates a phaseshift to beta-sheet form;

FIG. 22 is a conceptual illustration of a comparison of where Seq. Id.No. 7 and Seq. Id. No. 8 overlap in structure;

FIG. 23 is a graph of experimental results showing that peptides canself-associate;

FIG. 24 is a graph of fluorescence data showing the efficiency ofexcimer formation under low concentrations;

FIG. 25 is a graph of fluorescence experimental results showing theeffect of nuclei on self-association due to catalytic conformationaltransition;

FIG. 26 contains two graphs of fluorescence experimental results showingthe interaction of Seq. Id. No. 7 and Seq. Id. No. 8 at differentratios; wherein FIG. 26 a on the left shows a 1:1 mixture and FIG. 26 bon the right shows a 100:1 mixture;

FIG. 27 contains four graphs of fluorescence experimental resultsshowing the effect of nuclei on self-association. FIGS. 27 a, b, c and dshow the results at 24 hours, 48 hours, 144 hours and 336 hours,respectively;

FIG. 28 is a graph of fluorescence experimental results showing theeffect of nuclei on self-association due to catalytic conformationaltransition at 1 hour in FIG. 28 a on the left and at 150 hours in FIG.28 b on the right;

FIG. 29 shows peptide Seq. Id. No. 9, which is used to form sequencesfor a generalized dendrimer structure of this invention;

FIG. 30 shows a peptide sequence, i.e., Seq. Id. No. 10, for a preferredembodiment of a specific dendrimer structure of this invention;

FIG. 31 is a conceptual diagram of an experimental device; and

FIG. 32 is a system diagram of preferred embodiments of the invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

It is to be understood that the invention is not limited to the examplesdescribed herein. All technical and scientific terms used herein havemeanings as commonly understood by one of ordinary skill in the artunless otherwise defined. All publications referred to herein are whollyincorporated by reference to describe methods and materials forimplementing aspects of the invention.

The present invention detects the presence of abnormal proteins andproteinaceous particles based on a method that utilizes catalyticpropagation. Upon interaction of a sample, containing abnormal proteinsor proteinaceous particles, with a peptide probe of the invention, thepeptide probe undergoes conformational changes resulting in theformation of aggregates. The addition of the abnormal proteins andproteinaceous particles catalyzes the formation of the aggregates andcauses further propagation of this conformational transition. Theresulting aggregates are then easily detected using common analyticalinstrumentation and techniques.

The abnormal proteins and proteinaceous particles on which the inventionfocuses are proteins, protein based chemical structures such as prionsand protein subunits such as peptides that are capable of conformationalchanges that lead to the formation of aggregates and ultimately todisease states.

These proteins and proteinaceous particles form aggregates by shiftingfrom a monomeric to a multimeric state. The shift from one distinctstate to the other requires a driving force that is commensurate withthe energetic difference between the two conformational states as shownin FIG. 7.

A preferred example of such proteinaceous particles is that of a prionprotein. Prions can exist in one of two distinct conformationscharacterized by having a secondary protein structure that is eitherpredominately alpha-helical or predominately beta-sheet; where thepredominately beta-sheet conformation has a much higher preference toexist in a multimeric state. As a result, predominately beta-sheet (orbeta rich) secondary structure is more typical of abnormally folded ordisease-causing proteinaceous particles since their preference toaggregate is likely to be disruptive in an in vivo environment.

FIG. 1 shows illustrations of both the alpha-helical monomer 10 and thebeta-sheet dimer 12 forms of a TSE conformer (or alternative secondarystructure). Research has shown that the normal wild-type (wt) form ofprion protein (PrP^(c)) prefers a monomeric state, while the abnormal,disease-causing form (PrP^(Sc)) more readily takes on a multimericstate.⁴ ⁴ Fred E. Cohen, et al., Pathologic Conformations of PrionProteins (Annu. Rev. Biochem. 1998) 67: 793-819.

This distinction between the secondary structure of the normal form ofprion protein and the abnormal form as well as its propensity to causeaggregation is exploited in the present invention to allow detection ofthe abnormal form even in samples with very low levels of infectiousabnormal protein.

The mechanism of the invention is shown in a schematic in FIG. 2. Thetop row of the schematic shows an example of an unknown sample of TSEprotein 20 represented as containing aggregated beta-sheets 12. Thebeta-sheets are then disaggregated 22 by subjecting the sample tocommonly known disaggregation methods such as sonication. This isfollowed by the addition of labeled peptide probes 14 which are allowedto bind to the sample 20. Presence of the beta-sheet conformation in thesample 20 induces the peptide probes to also shift to beta-sheetformation 16. In this manner the transition to beta-sheet is propagatedamong the peptide probes 14 thereby causing new aggregates 18 to form.The resulting transition to a predominately beta-sheet form andamplified aggregate formation can then easily be detected using commonanalytical techniques such as light scattering and circular dichroism(CD); and in a particularly preferred embodiment where the peptide probeis fluorescent labeled, fluorescence detection instrumentation can alsobe used.

The bottom row of FIG. 2 shows an alternative example in which theunknown sample of TSE protein 20 is represented in its normalalpha-helical form 10. For consistency, the sample is subjected to thesame disaggregation process described above. Upon addition of thelabeled peptide probes 14, neither a transition to beta-sheet form norbinding to the unknown samples occurs. As a result, there is noaggregate fluorescence signal in the case of a labeled peptide probe aswell as no detection of aggregate formation by other analytical tools.Based on this schematic, unknown samples can be tested for the presenceor absence of such abnormal protein conformations or sequences.

A preferred embodiment of the invention involves the following basicprocedures. Peptide probes 14 are selected in order to be added to anunknown or test sample 20 at a later stage in the process. The peptideprobes 14 are preferably proteins or peptide sequences that havesecondary structures of predominately alpha-helix or random coil. In aparticularly preferred embodiment, the peptide probes 14 are peptidefragments consisting of a helix-loop-helix structure as found in lysine.In another particularly preferred embodiment, the peptide probes can bemade of a peptide sequence chosen from wild-type (wt) TSE, from adesired species-specific TSE peptide sequence, or even from aselectively mutated TSE sequence that has been mutated in such a manneras to render it destabilized and noninfectious. Additionally, extrinsicfluors such as pyrene can be added or designed into the peptide probe toallow detection of anticipated conformational changes using commonfluorescence detection techniques.

Once a peptide probe 14 is selected, it is added to a test sample 20.Prior to the addition of the peptide probe 14, however, it is preferredto have the sample 20 subjected to disaggregation techniques commonlyknown in the art, such as sonication. The disaggregation step allows anypotentially aggregated sample material 20 to break apart so that thesedisaggregated sample materials 22 are more free to recombine with thenewly introduced peptide probes 14; thereby facilitating the anticipatedcatalytic propagation.

After the test sample 20 or disaggregated test sample 22 is allowed tointeract with the peptide probes 14. The resulting mixture is thensubjected to analytical methods commonly known in the art for thedetection of aggregates and to fluorescence measurements in cases wherefluorescent peptide probes 14 are used.

Unknown or test samples 20 containing any dominant beta-sheet formationcharacteristic of abnormally folded or disease-causing proteins resultsin an increase in beta-sheet formation and consequently aggregateformation in the final mixture containing both the test sample 20 andthe peptide probes 14. Conversely, unknown or test samples 20 which lacka predominantly beta-sheet secondary structure will neither catalyze atransition to beta-sheet structure 16 nor will propagate the formationof aggregates 18.

One of ordinary skill in the art can appreciate that the

means by which the initial conformational change can be triggered in thetest samples 20 can be varied as described in the following examples.The binding of a metal ligand could direct a change in the proteinscaffolding and favor aggregation. The expression or cleavage ofdifferent peptide sequences can promote advanced aggregation leading tofibril and plaque formation. Genetic point mutations can also alter therelative energy levels required of the two distinct conformations,resulting in midpoint shifts in structural transitions. Furthermore, anincrease in concentration levels could be sufficient to favor theconformational transition. Regardless of the initial trigger mechanism,however, the disease process in many of the abnormal proteinconformations such as in prion-related diseases always involves thecatalytic propagation of the abnormal conformation, resulting intransformation of the previously normal protein.

One of ordinary skill in the art can also appreciate that there are manycommon protein aggregate detection techniques many of which are based onoptical measurements. These optical detection techniques include, butare not limited to, light scattering, or hydrophobicity detection usingextrinsic fluors such as 1-anilino-8-napthalene sulfonate (ANS) or CongoRed stain, fluorescence proximity probes on the peptide fragments,including fluorescence resonance energy transfer (FRET) & quenching ofintrinsic tryptophan fluorescence through either conformational changeof monomer or binding at interface in alpha-beta heterodimer; theN-terminal loop region is particularly interesting in this regardselective binding to target protein, circular dichroism (CD) monitoringof actual conformation, nuclear magnetic resonance (NMR). Otherdetection techniques include equilibrium ultracentrifugation orsize-exclusion chromatography at the aggregation stage as well as otherstructural techniques. Examples and explanations of these methods can befound in Freifelder, David. Physical Biochemistry: Applications toBiochemistry and Molecular Biology, (W. H. Freeman Press, New York, 2nded. 1982). and in Copeland, Robert. Analytical Methods for Proteins,(American Chemical Society Short Courses 1994). both of which are whollyincorporated herein as prior art. Many of these enumerated optical andstructural methods are rapid, cost-effective and accurate.

Experiments were performed using model systems to show theconformational changes involved in the transition from a predominatelyalpha-helix to a beta-rich form. The model systems chosen used readilyavailable, nonneurotoxic polyamino acids such as polylysine andpolyglutamine. The polyamino acids were chosen because of theiravailability and more importantly because they are safe to handle thuseliminating the need for special handling or donning cumbersome extraprotective gear.

FIG. 3 shows a circular dichroism graph of experimentation withpoly-L-lysine 20 micro Molar (μM) 52,000 molecular weight (MW) as apeptide probe. The resulting graphs show:

-   -   Sample 24 which was maintained at pH7, 25° C. resulting in a        minimum at approximately 205 namometers (nm) indicating random        coil structure.    -   Sample 26 which was maintained at pH11, 50° C. resulting in a        minimum at approximately 216 namometers (nm) indicating        beta-sheet structure.    -   Sample 28 which was a 1:1 combination of samples maintained at        pH7, 25° C. and at pH11, 50° C. resulting in a minimum at        approximately 216 namometers (nm) indicating beta-sheet        structure.    -   Sample 30 which was a 1:1 combination of samples maintained at        pH7, 50° C. and at pH11, 50° C. resulting in a minimum at        approximately 216 namometers (nm) indicating beta-sheet        structure.

FIG. 4 shows an absorbance graph of experimentation with poly-L-lysine70 mircomolar (μM) 52,000 molecular weight (MW) as a peptide probe. Theresulting graphs show:

-   -   Sample 32 which was maintained at pH 11, 25° C. resulting in a        plateau at approximately 0.12 indicating predominately        alpha-helical structure.    -   Sample 34 which was maintained at pH7, 50° C. resulting in a        plateau at approximately 0.22 indicating random coil structure.    -   Sample 36 which was a 10:1 combination of samples maintained at        pH7, 50° C. and at pH11, 50° C. resulting in a steeper incline        from approximately 0.22 to 0.33 indicating an accelerated        transition from random coil to beta-sheet structure.    -   Sample 38 which was a 10:1 combination of samples maintained at        pH7, 25° C. and at ph11, 50° C. resulting in a gradual incline        from approximately 0.22 to 0.26 indicating a transition from        random coil to beta-sheet structure.

FIG. 5 shows general circular dichroism results of experimentation withpoly-L-lysine at varying temperatures and pH indicating its potentialfor transitioning from random coil to beta-sheet under the varyingenvironmental conditions. The results indicate that both temperature andpH play an important role in the transition.

The observations based on all of the modeling experimentation describedabove show that the addition of a relatively small amount of beta-sheetpeptide to random coil sample can result in a shift towards a beta-richconformation and such changes can be accelerated depending on thetemperature and pH environment of the samples.

FIG. 6 shows experimentation results using pyrene as a fluorescent probein proximal and distal locations in an alpha helical bundle structureundergoing conformational change. The pyrene excimer formation 15 isshown at 480 nm 42 and the spectra for a predominately alpha-helicalstructure 17 is contrasted 40 as well. Those skilled in the art wouldappreciate that other fluorescent probes such as FITC can also be used.

A primary objective of this invention also encompasses use of thecatalytic propagation of conformational change to directly correlate themeasures of abnormal prion presence with levels of infectivity. For thisreason we favor implementation of the invention in a manner where thereis no increase in resulting infectious products as a result of thepropagation. This can be achieved by placing a “break” in the linksbetween the chain of infection, transmission and propagation of theabnormal form. Such a “break” must occur at the transitional stagebetween the dimer and multimer forms of the aggregate. The physicalformation of the multimer form can be blocked by simply impeding thestep which leads to its formation. This may be done, preferably by usinga large pendant probe or by a neutral “blocker” segment, bearing in mindthat probes on linkers or “tethers” are more likely to encounter eachother and thus result in amplifying the signal.

In a particularly preferred embodiment of the invention, the peptideprobes 14 function in the manner described above. The peptide probes actas “nuclei”; wherein once the peptide probe 14 binds to a test sample20, or a sample known to have beta-rich structure 12, it is converted toa peptide probe conformer 16 which has the capacity to act as a triggerto bind to another peptide probe 14 and continues to induce the sameconformational change. Propagation of this reaction can then becontrolled by the peptide sequence chosen for the peptide probe 14 andby the experimental conditions. Thus, in situations where infectiouslevels are low and there is a need to amplify any existing abnormalproteinaceous particles in an unknown sample 20, it is preferred that apeptide probe 14 capable of rapid and continuous propagation of thereaction be chosen with which to nucleate the unknown sample 20. On theother hand, in situations where it is desired to correlate detection ofabnormally folded proteinaceous particles with levels of infectivity, itis preferred that peptide probe 14 chosen is one that is less likely toaggregate.

When more than one beta units come together, they act as nuclei toattract and stabilize other transient elements of secondary structure.See Stryer, Lubert. Biochemsitry. W. H. Freeman Press. (3rd ed. NY 1988)p 35. In choosing the peptide probe 14 with which to nucleate thisreaction there are several considerations to be made. Associations ofpeptide can be controlled by the thermodynamics of the solution in whichthey are in and by the presence of amorphous nuclei whichself-associate, crystalline nuclei which readily aggregate, specificpeptide sequences which may aggregate, but may do so under lowconcentrations which are difficult to measure by conventional means, orlarger peptide sequences modeled after known beta-sheet structures orproteins such as a beta-rich prion protein.

To demonstrate this embodiment of the invention, two peptide sequenceswere synthesized to be used as peptide probes 14. The peptide sequenceswere modeled after known prion protein (PrP) sequences shown in FIG. 13.The sequences in FIG. 13 correspond to binding regions that are verysimilar among the species shown. FIG. 14 shows the peptide sequences ofthe two synthesized peptides. The 19-mer sequence referred to as Seq.Id. No. 7 is closely modeled after residues 104 through 122 of the humansequence. The 14-mer sequence referred to as Seq. Id. No. 8 is closelymodeled after residues 109 through 122 of the human PrP sequence. Thesynthetic peptide probes 14 were also prepared with and without pyrenebutyric acid as a fluorescence marker.

Many experiments were performed to study the properties of the syntheticpeptides. Experiments were performed using analytical techniques commonin the art such as absorbance, fluorescence under varying excitation andexcitation of fluorescence. The peptides were studied at severalconcentrations ranging from 1 to 100 micro Molar (μM) and under varyingbuffer concentrations, pH, temperatures and ionic strengths.

FIG. 15 shows a graph of fluorescence-spectra results at differentpeptide concentrations. The data were collected over times ranging formone hour to one week with no experimental changes observed aftertwenty-four hours. The resulting graphs show:

-   -   Sample 46 which was at a concentration of 5 μM with a relative        fluorescence peak at approximately 0.1.    -   Sample 48 which was at a concentration of 10 μM with a relative        fluorescence peak at approximately 0.4.    -   Sample 50 which was at a concentration of 150 μM with a relative        fluorescence peak at approximately 4.7.        Note: data were also collected for Sample 52 at a high        concentration of 800 μM, but is not shown in the figure.

FIG. 16 shows a graph of the fluorescence spectra for samples 46 through52 normalized to the intensity at 378 nm for the initial scan. It wasobserved that the spectrum for Sample 52 which contained the highestpeptide concentration was markedly different leading to the conclusionthat there is excimer emission with a maximum at approximately 460 nm.

FIG. 17 is a graph of experimental results showing pyrene's excitationof fluorescence. The experiments were performed with excitationwavelengths at 365 nm to observe excimer emission at approximately 460nm. The excitation at 348 nm, however, increases the fluorescence signalby over a hundred times with no other modifications or signalamplification. To confirm that the pyrene conjugate was responsible forboth the major 398 nm emission as well as the one at approximately 460nm, the excitation spectra for fluorescence at 398 nm and atapproximately 460 nm were recorded and are shown in FIG. 18. Both theexcitation spectra are nearly identical with a 365 nm maximum confirmingthat emission at approximately 460 nm is associated with the formationof excimers by two pyrene groups as in the following.Pyr*+Pyr=(Pyr _(—) Pyr)*where Pyr is a pyrene molecule and Pyr* is a pyrene in its excited form;the (Pyr_Pyr)* represents the formation of excited dimer. More generalinformation on excimers can be found in Freifelder, David. PhysicalBiochemistry: Applications to Biochemistry and Molecular Biology, (W. H.Freeman Press, New York, 2nd ed. 1982), at 559.

Experiments were also performed to study the stability of the peptides.FIG. 19 shows experimental data obtained from circular dichroism (CD)analysis of the 19-mer under different condition. The CD spectra wererecorded for a number of peptide concentrations ranging from 20 to 100mM. The results show that the 19-mer is largely coiled and exhibits highthermodynamic stability under the experimental conditions tested such asvarying pH, ionic strength and temperature. As expected, the addition oforganics such as acetonitrile and trifluoroethylene (TFE) encourage theformation of the secondary structure. FIG. 20 shows both the previousresults and the results of a similar experiment in which the 19-mer wasmixed with its shorter analog, the 14-mer. In this experiment, the19-mer and 14-mer were combined 100:1 for one hour and assembled underdilute conditions in the micro molar range. Sample curves 60 through 64which correspond to the mixture showed that the mixture of the oligomerssignificantly differed from the CD spectra of sample curves 52 through58 which represent the 19-mer alone, indicating strong interactionsbetween the mixed molecules. As a result, the 14-mer triggersconformational changes in a peptide probe 14 made of the 19-mer.

In a paper published by Prusiner, et al., CD data show that the Seq. Id.No. 7, 19-mer exhibits coil-like conformation, whereas the Seq. Id. No.8, 14-mer is largely beta-sheet as shown in FIG. 21 a for a 3 mMconcentration sample from the paper. The 19-mer, however, can betransformed from its coil-like conformation to a beta-sheet conformationthrough interaction with a very small fraction of the 14-mer as shown inFIG. 21 b which was tracked over a twenty four hour time period. SeePrusiner, et al. Prion protein peptides induce alpha-helix to beta-sheeconformational transitions. Biochemistry. 34:4186-92.

FIG. 22 shows a conceptual figure of the secondary structure of the twosynthetic peptides (where C=coil and H=helix) based on the applicationof various secondary structure algorithms to the sequences of both ofthe synthetic peptides. The resulting projection, however, does notentirely agree with the CD results. Based on the CD results, theconformations of both synthetic peptides are clearly concentrationdependent. Moreover, while the 19-mer exhibits largely a coilconformation that is fairly stable under a wide variety of theexperimental conditions tested, the 14-mer exhibits a transition fromcoil or hairpin to beta-sheet structure depending on its concentration.

More experiments were performed to determine if the 19-mer couldself-associate. FIG. 23 shows a graph of fluorescence results showingthat the 19-mer could self-associate with increasing concentration asshown in Sample curve 66 and at low concentrations with pH modificationsto give a net neutral charge while using potassium chloride (KCl) toscreen the charge as shown in Sample curve 68. The 19-mer can alsoself-associate at low concentrations with the introduction of some typeof nucleating agent, as discussed earlier. Thus, the conditions forself-association can be optimized to adapt to a desired type ofdetection.

The same samples; Sample curve 66 containing 0.1 M TRIS buffer at pH 6to 9 and Sample curve 68 containing 0.1 M TRIS buffer at pH 10 to 11 inthe presence of KCl at 100 to 500 mM, are shown again in FIG. 24 toreflect the efficiency of excimer formation under low concentrations.The ratio of the fluorescence intensities as measured at 378 nm (I_(M))and at 460 nm (I_(E)) was chosen to monitor the self-association as afunction of the peptide concentration at 25° C. It was also shown thatscreening of the electro-static interactions (pI=10) encouragedself-association at extremely low concentrations on the order of lessthan 10 micro Molar.

In order to further study the effect of nuclei on the self-associationof the 19-mer, more fluorescence measurements were taken of 19-mer insolution nucleating with small amounts of already self-associated 19-merunits. The sample solutions range from concentrations of 200 to 800micro Molar and are described in FIG. 25. The kinetics of association indilute solutions of 20 micro Molar were also monitored.

FIG. 26 a shows more fluorescence data of the 19-mer in water 70,acetonitrile 72 and TFE 74 after twenty-four hours. FIG. 26 b shows theexperimental results for a 100:1 combination of the 19-mer and 14-mer inwater 76, acetonitrile 78 and TFE 80 after twenty-four hours. In both ofthe graphs in FIG. 26 peptide association was monitored by theappearance of excimer emission at approximately 460 nm.

FIGS. 27 a, b, c, and d show four fluorescence data graphs taken at 24,48, 144 and 336 hours, respectively. The measurements were taken todetermine the effect of pH, temperature, ionic strength, and organicadditives on the kinetics of the peptide associations studied for the19-mer model peptide. The fluorescence intensities as measured at 378 nmfor monomeric units and 460 nm for associations were used tocharacterize the I_(E)/I_(M) ratio or self-association of the peptide.

Additional fluorescence results are shown in FIG. 28 where an insolublefraction of the peptide was extracted and dissolved in organic solventcontaining methanol/ethanol/dimethylformanide and then analyzed.Fluorescence detection results of the “insoluble” portion show highlevels of peptide association wherein the I_(E)/I_(M) ratio equals 2. Asmall aliquot of “insoluble” portion was added to nucleate 20 microMolar 19-mer peptide solutions which were then analyzed and are reportedin the same graph. The results show that the presence of the nucleatingfraction significantly increased the efficiency of the peptideassociation and this can be seen more dramatically in FIG. 28 b at 150hours.

The observations of these experiments led to some of the followingconclusions.

-   -   Fluorescence of pyrene, which is covalently attached to the        peptide probe 14 in preferred embodiments, allows monitoring of        peptide self-association in this model system. It can also be        used as an index of conformational change and especially since        at low concentrations, the peptide association is difficult to        measure using nonoptical techniques.    -   The fluorescence data shows that self-association of the Seq.        Id. No 7, 19-mer, can be promoted by adjusting ionic strength or        pH.    -   The fluorescence data also shows that the kinetics of the        conformational changes can be modulated by controlling solvent        parameters and the peptide probe sequence.    -   The kinetics of the self-assembly or association process can be        controlled or regulated by the addition of or by preexisting        nucleating associated forms. This strongly supports the        conclusions that the conformational transitions of the 19-mer        can be autocatalytic.

In a particularly preferred embodiment, the peptide probes 14 can beused to detect proteinaceous particles such as in prion-like structuresexhibiting coil to beta-sheet transition. According to Prusiner, et al.Prion protein peptides induce alpha-helix to beta-sheet conformationaltransitions. Biochemsitry. 34:4186-92 (1995). As a result, syntheticpeptide probes such as the Seq. Id. No 7, 19-mer should beconformationally sensitive to the presence of prion-like substances thatundergo this conformational shift. Moreover, because an intrinsicoptical reporter, such as pyrene can be added to the peptide probe, thisembodiment of the invention has the added advantage of being able todetect such prion-like substances in test samples 20 such as blood,lymph, CSF and even tissues other than brain homogenate that typicallycontain very low levels of abnormal prion substances that are otherwisetoo difficult to detect. The intrinsic optical reporter allows optical(fluorescence) measurements to be taken of the peptide probe associatesthat form upon interaction with nucleating samples such as an abnormalprion.

In another particularly preferred embodiment of the invention, thepeptide probes 14 are synthesized based on the structure of a dendrimer;dendrimers being synthesized three-dimensional highly branchedmacromolecules. The advantages of using a dendrimer probe 15 aremultifold. Dendrimers should increase the speed of the assay kineticsthereby relaying quicker test results. This can be especiallyadvantageous in assembly line applications of the invention whereproducts or specimens in mass quantities can be quickly tested for thepresence of abnormal proteinaceous particles. This embodiment is alsoextremely beneficial in applications where quick decisions must be basedon the detection results. This embodiment is also advantageous for usein these applications as well as others since the highly branchedstructure of the dendrimer prevents amplification of abnormalproteinaceous particles or aggregates. By preventing such amplificationof the abnormal particles, it becomes very simple to correlate thedetection results with the level of abnormal aggregates existing in atest sample 20. Furthermore, it is also safe to handle since thesynthetic probe itself is nonneurotoxic and amplifies signal withoutamplification of any highly infectious particles that may be preexistingin a test sample 20. Thus, it eliminates the need for extra precautionsor sterilization in many of the steps of the assay method.

A generalized dendrimer 15 structure is shown in FIG. 29 and is referredto as Seq. Id. No. 9. In a particularly preferred embodiment of theinvention, a specific dendrimer structure was designed and synthesized,referred to as Seq. Id. No 10, and is shown in FIG. 30.

In FIG. 30, the specific dendrimer structure is basically aloop-turn-loop structure as illustrated by FIG. 30 a. In FIG. 30 b, itis shown that the sequence is modeled after the human PrP sequence shownin FIG. 14 in residues 126 through 104 plus 109 through 126. Thisstructure shows the region on the right 74 as an inverted form of thePrP sequence. This was done to take advantage of the five aminoacidswhich naturally form a loop in order to place hydrophobic pyrene in acorresponding hydrophobic region. Also the valine-valine fragment isessential to beta-sheet formation and so is retained in the sequence. Inthe figure, green denotes possible mouse variants. The amyloidogenicpalindrome region 70 may be changed to SS or SSS/AAA. The central region72 is a loop sequence with steric constraints, thus it is possible toadd tryptophan for steric and fluorescence considerations.

Modifications of the aminoacid sequence such as one or more deletions orinsertions are possible as alluded to above, provided that the dendrimerretains its branched loop-turn-loop structure as well as aminoacidsessential to beta-sheet formation, and preferably contains an opticalreporter.

FIG. 10 shows a schematic diagram of how the dendrimer probes 15 amplifysignal and propagate conformational change without aggregation andwithout increasing the biohazard or infectious nature of an abnormalprotein or prion test sample 12. The figure shows that once thedendrimer probes 15 come into contact with the abnormal sample 12, thedendrimer probe 15 undergoes the conformational shift to a predominatelybeta-sheet structure 17. The newly formed beta-rich dendrimer probe 17nucleates other dendrimer probes 15 to make the same transition. Bydoing so, any optical signal associated with the dendrimer probe 15 isamplified as more probes 15 shift to the beta-rich state 17.

It is important to note that the minimal detectable concentration ofpyrene only provides a number for the peptide probe 14 concentrationthat can be worked with; but the detection limit of the assay is notdependent on it because it is the resultant of the fluorescent ensemblethat is being observed. In other words, the real measurement of interestand the rate limiting step in the analysis is the amount of abnormal e.g. prion protein that need to be present in the sample 20 to initiate aconformer change in the peptide probe 14. Immunoassays are typicallysensitive in the picomolar range. Nevertheless, once the conformerchange is initiated in a single peptide probe 14, the catalyticpropagation of its beta-rich structure allows detection in samplespreviously considered to have abnormal particles 12 at concentrationstoo low to detect.

Due to its ability to safely, quickly and noninvasively detect abnormalproteinaceous particles such as misfolded proteins, prions, aggregatesand fibrils that may lead to toxic plaque formations, the method of thisinvention is widely applicable to many industries. By example, some ofthose industries include the diagnostics markets in animal health andhuman health, the food industry, pharmaceutics, especially for screeninganimal by-products, transplant/transfusion and vaccine supplies,research and development in such areas as chemotherapies for TSE's, aswell as national security in the area of biosensors for biowarfareagents.

Accordingly, in yet another preferred embodiment of the invention, themethods discussed herein can be applied for use with a simple detectioninstrument such as the one shown in FIG. 31. The device shown in FIG. 31is a simple optical device that includes a light source 80 shown in bluee. g. lamp or laser; a T-format sample cell 82 shown in grey; and aphotomultiplier tube 84 shown in pink. In certain applications it may bedesirable to have the method distributed as an assay that includes sucha simple device.

Accordingly, the present invention is not limited to the specificembodiments illustrated herein. Those skilled in the art will recognize,or be able to ascertain that the embodiments identified herein andequivalents thereof require no more than routine experimentation, all ofwhich are intended to be encompassed by claims.

Furthermore, it will be understood that the foregoing disclosure isintended to be merely exemplary, and not to limit the scope of theinvention—which is to be determined by reference to the appended claims.

1. An in vitro method for detecting a target protein having apredominantly β-sheet secondary structure, comprising: forming a mixtureby adding a propagation catalyst to a sample suspected of containing atarget protein having a predominantly β-sheet secondary structure,wherein the propagation catalyst is a peptide that: (i) has apredominantly alpha-helix and/or random coil secondary structure andinteracts with protein having a predominantly β-sheet secondarystructure; (ii) undergoes a conformational shift that results in adecrease in alpha-helix and/or random coil secondary structure and anincrease in beta-sheet secondary structure upon contact with proteinhaving a predominantly β-sheet secondary structure or upon contact withanother such propagation catalyst that has undergone such aconformational shift; and (iii) is labeled with pyrene; allowing thepropagation catalyst and any target protein present in the sample tointeract; and detecting any increase in beta-sheet secondary structurein the mixture by detecting pyrene excimer formation, the increase beingdue, at least in part, to an increase in beta-sheet secondary structureof the propagation catalyst, wherein any such increase indicates thepresence of target protein in the sample.
 2. The method of claim 1,wherein said propagation catalyst is labeled with a pyrene label at eachof its N-terminus and its C-terminus.
 3. The method of claim 2, whereinwhen said propagation catalyst undergoes said conformational shift,interaction between the pyrene label at each of its N-terminus and itsC-terminus results in pyrene excimer formation.
 4. The method of claim1, further comprising adjusting a reaction condition to increase ordecrease pyrene excimer formation.
 5. The method of claim 4, whereinsaid reaction condition is selected from the group consisting of: ionicstrength of the sample, pH of the sample, concentration of the sample,temperature, and the presence or absence of nucleating agents.
 6. Themethod of claim 1, further comprising modifying the amino acid sequenceof the propagation catalyst to increase or decrease pyrene excimerformation.
 7. The method of claim 1, wherein the method furthercomprises, prior to the step of adding the propagation catalyst to thesample, the step of subjecting the sample to a disaggregation technique.8. The method of claim 1, wherein said detecting step comprisesdetecting aggregates comprising the propagation catalyst.
 9. The methodof claim 1, wherein said target protein is associated with a disease.10. The method of claim 9, wherein said disease is selected from thegroup consisting of Alzheimer's Disease, Huntington's Disease, andprion-associated diseases.
 11. The method of claim 10, wherein saidtarget protein is selected from the group consisting of Aβ protein,huntingtin protein, transmissible spongiform, and prion proteins. 12.The method of claim 1, wherein said sample comprises a biological samplefrom a subject.
 13. The method of claim 1, wherein said sample comprisesa biological sample from a living subject.
 14. The method of claim 1,wherein said sample comprises a biological sample from a human subject.15. The method of claim 1, wherein said sample comprises blood, lymph,CSF, or tissue.
 16. The method of claim 1, wherein said sample comprisesa biological sample and a solvent.